Over the past two decades, various polymer-based non-viral vectors for gene delivery have been developed by structure design and formulation. Most of the non-viral vectors currently developed are polycations. Among the polycations investigated, high molecular weight polyethylenimine (HPEI) has demonstrated a higher gene transfection efficiency in various types of cells both in vitro and in vivo. This attribute has contributed to its use to condense plasmid DNA effectively into colloidal particles via its proton-sponge effect. However, its non-degradability, aggregation, cytotoxicity and/or short-circulation time in vivo have limited its clinical applications. Modifications of HPEI to reduce cytotoxicity, while retaining its high potential to bind and condense plasmid DNA for gene delivery have been investigated previously, but with limited success. One effort has been to modify HPEI (25 k) with hydrophilic polyethylene glycol (PEG) aiming to primarily shield the surface charges of the PEI, thus reducing toxicity. However, this modification led to reduced transfection activity when compared to HPEI alone. Williams et al. synthesized a PEG550-PEI 2000 copolymer and examined the efficiency of the polymer for the delivery of 2′-O-methyl-phosphorothioate antisense oligonucleotide (2′-OMePS) in tibialis anterior (TA) muscle of mdx mice for Exon 23 skipping, but the efficiency was still limited and probably due to its hydrophilicity. Amphiphilies pluronic (such as F127, SP1017) have been evaluated and found effective in enhancing gene transfection efficiency of naked DNA or antisense oligonucleotides in skeletal muscle (Lu, Q L, Bou-Gharios, G and Partridge, T A. (2003). Non-viral gene delivery in skeletal muscle: a protein factory. Gene Ther 2003, 10, 131-142; Lemieux, P, Guerin, N, Paradis, G, Proulx, R, Chistyakova, L, Kabanov, A et al. (2000). A combination of poloxamers increases gene expression of plasmid DNA in skeletal muscle. Gene Ther 2000, 7, 986-991; Pitard, B, Pollard, H, Agbulut, O, Lambert, O, Vilquin, J T, Cherel, Y et al. (2002). A nonionic amphiphile agent promotes gene delivery in vivo to skeletal and cardiac muscles. Hum Gene Ther 2002, 13, 1767-1775; Cho, K C, Choi, S H and Park, T G. (2006). Low molecular weight PEI conjugated pluronic copolymer: Useful additive for enhancing gene transfection efficiency. Macromol Res. 2006, 14, 348-353).
Applicants have developed cationic amphiphilic polymers constructed from poloxamers/pluronic and polyamines and achieved considerable enhancement for plasmid DNA (pDNA) or antisense oligonucleotides (AOs) delivery in vitro and in vivo (Wang M X, Lu P J, Wu B, Tucker J D, Cloer C and Lu Q L. High efficiency and low toxicity of polyethyleneimine modified pluronics (PEI-Pluronic) as gene delivery carriers in cell culture and dystrophic mdx mice. J Mater Chem 2012; 22: 6038-6046; Wang M X, Wu B, Lu P J, Tucker J D, Cloer C and Lu Q L. Polyethylenimine Modified Pluronics (PCMs) Improve Morpholino Oligomers Delivery in Cell Culture and Dystrophic mdx Mice. Mol Ther 2013; 21: 210-216; Wang M X, Wu B, Tucker J D, Lu P J, Cloer C and Lu Q L. “Evaluation of Tris[2-(acryloyloxy)ethyl]isocyanurate Cross-linked Polyethylenimine as Antisense Morpholino Oligomer Delivery Vehicle in Cell Culture and Dystrophic mdx Mice”. Human Gene Therapy, 2014, 25(5), 419-427; Wang M X, Tucker J D, Lu P J, Wu B, Lu Q L. “Tris[2-(acryloyloxy)ethyl]isocyanurate cross-linked low-molecular-weight polyethylenimine as gene delivery carriers in cell culture and dystrophic mdx mice”, Bioconjugate Chemistry, 2012, 23, 837-845).
However, it is generally difficult to control the molecular size and composition of the polymers as well as the corresponding polymer-AO conjugates. This often results in poor reproducibility of copolymers and makes further optimization more difficult.
The success of gene/oligonucleotide therapies often relies on the ability of systems to deliver the therapeutic gene/oligonucleotides to the target tissue relatively efficiently and safely. Non-viral gene delivery systems, based on naked DNA/oligonucleotides, have advantages over viral vectors for simplicity of use and lack of specific immune response related to viral infection. However, naked DNA/oligonucleotides are generally difficult to deliver into target cells in vivo. An ideal non-viral gene carrier typically must condense DNA/oligonucleotides into small polyplexes, protect them from enzymatic degradation, penetrate cell membranes, and deliver their cargo into the nucleus efficiently without causing significant cytotoxicity. Consequently, a number of approaches have been proposed to develop cationic polymers with higher gene transfection efficiency and lower cytotoxicity. However, development and use of relatively effective and safe vectors for plasmid DNA (pDNA)/oligonucleotide delivery generally remain challenging, especially for treating muscle and other genetic disorders, which require systemic delivery.